The present invention relates to displacement chromatography and displacement chromatographic apparatus.
Chromatography is a method for separating components from a mixture of chemical substances. It is particularly useful with mixtures of compounds whose chemical or physical properties are so nearly identical as to make other separation techniques difficult or impractical. The various components are resolved by their selective retardation as they are transported through a bed of sorptive medium by a moving fluid. The separation of the components depends upon their relative affinity for the sorbent and the moving fluid, the length of the chromatographic device, and the fluid flow rate.
Four separate mechanisms or processes exist for retention of sample molecules by the stationary phase. These, in turn, give rise to four basic chromatographic methods, namely, liquid-liquid, liquid-solid, ion-exchange, and size-exclusion chromatography. Liquid-liquid or partition chromatography involves a liquid stationary phase whose composition is different from that of the liquid moving phase. Simple molecules distribute between the mobile and stationary liquid phases, just as in liquid-liquid extraction with a separatory funnel. The moving- and stationary-phase liquids must be immiscible. Liquid-solid or adsorption chromatography involves high-surface-area particles, with retention of sample molecules occurring by attraction to the surface of the particles. In ion-exchange chromatography, the stationary phase contains fixed ionic groups such as sulfonate (--SO.sub.3.sup.-) along with counter-ions of opposite charge, e.g., Na.sup.+. The counter-ions are also normally present in the mobile phase in the form of a dissociated salt, e.g., NaCl. Simple ionic species, e.g., X.sup.+, are retained by an ion exchange mechanism: EQU X.sup.+ +--SO.sub.3.sup.- Na.sup.+ .revreaction.Na.sup.+ +--SO.sub.3.sup.- X.sup.+
Finally, in size-exclusion or gel permeation chromatography the column packing is a porous material with pores of a certain size. Molecules that are too large are excluded from all the pores, whereas small molecules can penetrate most of the pores. Thus, very large molecules move through the column quickly and smaller molecules are retained by the packing. Usually, separation in size-exclusion chromatography is determined strictly by molecular size.
Two additional chromatographic methods result from modification of liquid-liquid chromatography, namely, bonded-phase chromatography and ion-pair chromatography. Bonded-phase chromatography uses an organic stationary phase that is chemically bonded to the particles in place of the mechanically held liquid phase used in liquid-liquid chromatography. Ion-pair chromatography can be regarded as a combination of liquid-liquid chromatography or bonded-phase chromatography and ion-exchange chromatography. Ion-pair chromatography can be carried out with either a mechanically held liquid stationary phase or a bonded phase.
Within the four basic chromatographic methods, at least two entirely different, distinct separation protocols exist. These have been named displacement chromatography and analytical or elution chromatography. In displacement chromatography at least two species B and C are sorbed onto the stationary phase or resin which was originally loaded with a third specie A having less affinity for the stationary phase than either B or C. Typically, up to 50 percent of the total capacity of the resin is loaded with species B and C. A displacer reagent consisting of a specie D is then passed through the resin. Specie D has an affinity for the resin greater than either B or C and causes the mixture of B and C (1) to be displaced from the resin, and (2) to segregate into bands of equal concentration that elute from the resin in a head-to-tail fashion.
In analytical or elution chromatography, the mixture to be separated is initially adsorbed on the stationary phase. An eluting species or eluent contained in the mobile, liquid phase is then passed through the resin. The eluent also has affinity for the components of the mixture which is different from the affinity of the components for the resin. The eluent need not have any affinity for the stationary resin phase. Separation is achieved by the competition of the stationary resin phase and the mobile eluent phase for the components in the mixture. The components are resolved into symmetrical Gaussian-shaped peaks that are separated on the resin to an extent determined by the resolution, selectivity, and efficiency of the system. The resin cannot be loaded to more than 1-5% of the total resin capacity with the mixture before resolution and peak symmetry are adversely affected.
Analytical or elution chromatography is designed to separate small quantities of a mixture to a very high degree of separation or resolution. It is therefore best suited and most widely used in small-scale analytical applications. It is not a method that lends itself readily to separation of large amounts of material.
Displacement chromatography, on the other hand, is a preparative technique that can separate 10 to 50 times the amount of material that can be processed by elution chromatography. Although resolution of the components is somewhat less, displacement chromatography is the method of choice for large-scale, industrial chromatographic separations.
In its conventional implementation, chromatography is a batch process. A volume of the mixture to be separated is introduced at the feed end of a chromatographic column (i.e., a single cylindrical tube filled with resin) and the components are resolved, in time, into individual bands along the column length. The separated components are then recovered at the column exit since they are eluted at different times.
Elution chromatography is applicable to many types of mixtures and has high resolution capabilities and high versatility, but its deficient throughput capacity and lack of continuous operation keep it from being a separation technique suitable for large scale, industrial operations. Attempts have been made to increase the capacity of chromatographic devices. One attempt is the continuous annular chromatography (CAC) system based upon elution and gradient elution chromatography.
Continuous chromatographic separations can be achieved by means of the CAC system. The CAC apparatus consists of an annular particulate bed having adsorbent particles packed in a space between two concentric cylinders. While the column assembly is slowly rotated about its axis, an eluent solution and a feed mixture are continuously fed to the annular bed. The eluent is uniformly fed to the entire circumference, but the feed mixture is introduced at only a fixed point on the circumference that remains stationary in space. As time progresses, helical component bands develop and separate from the feed point, with slopes dependent upon eluent velocity, rotational speed, and the distribution coefficient of the components. These bands are fixed in space and exit at separated, stationary exit points at the opposite end of the annular bed. As long as conditions remain constant, the angular position of each component band from the feed point also remains constant. No regeneration is required in CAC systems because the feed mixture is removed from the particulate bed by the eluent without changing the chemical make-up of the particulate bed. Hence, the CAC system is a continuous, steady-state process.
In the CAC system, the length and time coordinates characteristic of conventional chromatographic separation is replaced with the length and angle coordinates of the rotating bed. In this respect, conventional elution and gradient elution chromatography and elution and gradient elution CAC, respectively, are completely analogous. Therefore, elution and gradient elution CAC should be able to perform continuously any separation capable of being preferred by conventional batch elution and batch gradient elution chromatography.
Since only a small portion of the resin capacity, typically less than about 5 percent, can be loaded with the chromatographic species to be separated in both elution and gradient elution CAC, the CAC methodology nevertheless has a significant drawback in that it can only separate relatively small amounts of chromatographic species per unit volume of resin.